The continued spread of Staphylococcus aureus strains that are resistant to available antibiotics has created a severe problem for treating S. aureus infections. One of the critical impediments to developing drugs that are effective against these deadly MRSA strains is the lack of new antibiotic targets. The trans-translation pathway for resolving stalled ribosomes is a potential target for drug development because it is found in all bacteria and is required for growth of MRSA in culture. The long-term goal of this project is t identify specific inhibitors of trans-translation in S. aureus that can be used as lead compounds for drug development. The overall objective of this proposal is to validate trans-translation as a viable antibiotic target in S. aureus and to develop a primary assay compatible with high-throughput screening (HTS) and secondary assays that can be used to identify inhibitors of trans-translation in S. aureus. The rationale that underlies the proposed research is that validation of trans-translation in MRSA as a drug target and development of assays for HTS will lead to rapid identification of new antibiotics that will dramatically improve treatment for MRSA. The specific aims of this proposal are to confirm that tmRNA and SmpB are essential for S. aureus growth in culture and during infection, to develop primary and secondary screening assays to identify inhibitors of trans-translation in S. aureus, to conduct a pilot screen for tmRNA-SmpB inhibitors, and to use established ex vivo and in vivo assays to characterize hits from screening. Depletion strains and small molecule inhibitors will be used to remove trans-translation activity in during growth in culture and during infection. The results of these experiments will provide the basis for understanding the importance of trans-translation in S. aureus as well as determining if this pathway can be targeted for drug development. An established HTS assay and tmRNA-SmpB activity assays will be adapted for use with S. aureus components. Results from these studies will allow future HTS and high-level optimization of drug candidates. By targeting a pathway that has not been used for antibiotic development, this project will yield new compounds that can be used individually or in combination with existing MRSA therapies.